Rapid method of detection and enumeration of sulfide-producing bacteria in food products

ABSTRACT

A rapid method for detecting spoilage of a food sample, particularly a fish sample, by detecting and enumerating sulfide-producing bacteria (SPB). A growth medium containing iron and sulfur is combined with the food sample forming an incubation mixture which is incubated for a period of time. In one embodiment, a plurality of fluorescence measurements are taken during an incubation period of about 3 hours to 17 hours at 30° C. SPB are determined to be present in the sample if the fluorescence measurement initially increases and then decreases to form a fluorescence maximum (peak). The time to detection of the fluorescence peak can be used with a correlation schedule to enumerate the SPB in the food sample. In another embodiment, a visual test can also be used to identify color changes in the incubation mixture to provide a semi-quantitative enumeration of SPB effective after about 3 hours to 17 hours of incubation.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation-in-part of U.S. Ser. No.10/016,854, filed Dec. 14, 2001, now U.S. Pat. No. 6,632,632, theentirety of which is hereby expressly incorporated by reference hereinin its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND

The present invention relates to rapid methods for detection andquantification of microorganisms in food products for human consumptionor use, and more particularly to rapid methods for the detection andquantification of sulfide-producing bacteria in fish products.

Among the most predominant bacteria associated with spoilage of fishproducts, and food products in general are sulfide-producing bacteria(SPB) such as Shewanella putrefaciens. SPB are especially responsiblefor spoilage of many kinds of foods such as seafood, including fish,fish products, mussels, mussel products, shellfish and shellfishproducts and other meat products such as poultry (e.g., chicken andturkey), pork, beef, and lamb, and even dairy products such as cheese.SPB are particularly responsible for spoilage in fresh or cooledaerobically-packed seafood. These bacteria are present in seawater andon the surface of all living fish and shellfish, and are transferred tothe flesh during catch and processing. They grow to high levels andcause spoilage even when the fish are stored on ice (at approximately0-4° C.). The spoilage is mainly due to growth of psychrotropicbacteria, including S. putrefaciens.

Traditionally, microbial analysis of fish has been limited to totalviable counts. Indicator testing for total viable organisms andcoliforms are the most widely used tests for routine monitoring ofmicrobial contamination. However, during the last decade, it has beendiscovered that the shelf life of fish and shellfish is correlated tothe level of specific spoilage bacteria and not to the count of totalviable organisms. Spoilage is sensory detectable when the number ofsulfide producing bacteria exceeds 10⁷ colony forming units per gram(cfu/g) of fish muscle. In the case of cod from the North Atlantic, forexample, this level is reached after approximately 12 days when the fishare stored on ice, after approximately 7 days of storage at 4° C., andafter even shorter times when stored at higher temperatures. The growthof bacteria is exponential, and the shelf life of the fish can bepredicted from the number of SPB in the fish, and the growth rate ofthese bacteria at the actual storage temperature. In addition to sulfideproduction, SPB contribute to the accumulation of trimethylamine (TMA)in the fish. TMA is a primary component of unpleasant fishy odors.

Traditional agar plate methods have been developed for analysis of S.putrefaciens and other SPB in fish (Gram et al., 1987). In thosemethods, a non-specific growth medium is supplemented with thiosulfate,cysteine and ferricitrate. Bacteria that are able to produce sulfidefrom thiosulfate or cysteine appear as black colonies on the agar, dueto precipitation of ferrous sulfide. The detection is therefore directlyrelated to the spoilage property of the bacteria. This is a benefit inanalysis of spoilage bacteria, since the spoilage potential of themicrobe is more important that its identity. However, the agar platemethod has the same disadvantages as all other agar plate methods. Forinstance, it does not give the possibility for early warning and itrequires laboratory facilities, including an autoclave and techniciansskilled in sterile technique. The method is also time and laborintensive. Also, the lowest detection level is about 100 cfu/g, which isnot adequate for presence/absence tests. Furthermore, the requiredanalysis time is at least one to three days depending on the incubationtemperature. Since the analysis takes so long, this method of analysiscannot be used to make a timely decision regarding whether or not fishshould be purchased, sold, or used, or whether or not the fish can beused to produce other products. Therefore, more rapid methods ofdetection are desired to be able to expedite the decision-making processregarding how food products such as fish and shellfish which arecontaminated with sulfide-producing bacteria can be used, sold, orpurchased.

Other, more rapid, analyses based on bacterial growth with a possibilityfor early warning have been developed for total viable counts, forseveral hygienic indicator bacteria and for certain pathogens. Forexample, a sample is inoculated in a growth medium which is more or lessspecific for the bacterium that is going to be detected or quantified.The medium has an indicator that is a specific substrate for the desiredbacterium. The product produced by turnover of this substrate becomesdetectable (for example by fluorescence) when the number of microbesreaches a certain level. Other examples are impedimentary methods, wherethe indication is related to a change in the number of chargedmolecules. In these methods, the number of microbes in the sample iscalculated indirectly based on the time required until detection and onthe growth rate at the given conditions. However, all of these methodssuffer from one or more deficiencies, and there continues to be a needfor methods which will more rapidly detect and/or quantify SPB whichcause product spoilage.

SUMMARY OF THE INVENTION

The present invention comprises methods for rapidly detecting andquantifying sulfide-producing bacteria, for instance S. putrefaciens, ina sample of a food product comprising meat, dairy or fish. The foodsample is combined with a quantity of growth medium. The growth mediumpreferably comprises an iron compound (ferric citrate or ferroussulfate, for example) and organic and/or inorganic sulfur compounds(cysteine and sodium thiosulfate, for example) and forms an ironprecipitate (e.g., iron sulfide) when exposed to S. putrefaciens orother sulfide-producing bacteria. The growth medium and the sample formsan incubation mixture. The incubation mixture is incubated for apredetermined incubation period, for example from about four or fivehours to 16-18 hours.

The level of SPB in the sample is determined by using a visual detectionmethod to identify a color change, or by using fluorescence detectionmethods which detect trends in fluorescence production of the incubationmixtures which are correlated with SPB numbers.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings(s) will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 is a graph depicting results for rapid enumeration of S.putrefaciens in pure cell culture, wherein sample 1 (♦) has 3.9×10⁷cfu/ml of S. putrefaciens; sample 2 (▪) has 9.7×10⁶ cfu/ml of S.putrefaciens; sample 3 (▴) has 2.4×10⁶ cfu/ml of S. putrefaciens; sample4 (x) has 4.8×10⁴ of S. putrefaciens; sample 5 (∘) has 4.8×10³ cfu/ml ofS. putrefaciens and sample 6 (▬) has 3.8×10⁴ cfu/ml of of S.putrefaciens.

FIG. 2 is a graph showing the development of fluorescence in cod samplescontaminated with S. putrefaciens, wherein sample 1 (♦) has 1.4×10⁵cfu/ml of S. putrefaciens; sample 2 (▪) has 1.4×10⁴ cfu/ml; sample 3 (▴)has 1.4×10³ cfu/ml; sample 4 (●) has 1.4×10² cfu/ml; and sample 5 (▬)has 1.4×10¹ cfu/ml. The distance from the y-axis (start of the analysis)to the point where the fluorescence starts to increase is equal to the“time to detection” for S. putrefaciens.

FIG. 3 is a graph depicting the correlation (r²=⁻0.89, n=118) betweenthe time of detection (x-axis) and the number of naturally-occuringsulfide-producing bacteria in a variety of fresh fish productsdetermined by the standard method (y-axis), cultivated in IAL (Iron AgarLyngby) broth and incubated at 30° C.

FIG. 4 is a color chart for assessing color of an incubation mixtureafter an incubation period.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a rapid method for quantification ofsulfide-producing microbes, such as Shewanella putrefaciens in foodproducts, including meat, or diary, and particularly in fish andshellfish. The method is based on the formation of iron sulfide by SPBin a liquid growth medium. Iron sulfide formation is detected as achange in the background fluorescence in the growth medium or as a colorchange in the medium from yellow to gray to black or as a blackening onthe surface of an intact portion of the sample.

With the present invention it is possible to detect and enumerate evenlow numbers (<10⁴ cfu/g) of sulfide-producing bacteria in samples offresh fish (such as, but not limited to: cod (Gadus morhua), redfish(Norwegian haddock (Sebastus marinus)), salmon (Salmo salar), haddock(Melanogrammus aegifinus), coal fish (Pollachius virens) and wolf fish(Anarhichas lupus)) within, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 or 17 hours or increments there between hours, whichis considered sufficiently rapid and timely to allow corrective actions(e.g., to prevent or suspend the processing, purchasing, or selling) inthe case of unacceptable bacterial contamination in the food product. Italso is possible to use the method as a “pass-fail” test for SPB atparticular levels, when desired. The detection level is 1-5 bacteria pergram of sample, which is at least tenfold more sensitive than thedetection level in the traditional agar plate method.

As bacteria start to grow and produce H₂S, the properties of the mediachange as ferricitrate (or ferrous sulfate) is reduced and iron sulfide,FeS, is formed. When Fe³⁺ is reduced to Fe²⁺, the emission fluorescenceinitially increases, then decreases due to the darkening of the samplemixture caused by an increase in the amount of the iron sulfideprecipitate. This increase, then decrease, in fluorescence is expressedas a curve with a peak. Samples are read in fluorescent signal units(fsu), which is the relative intensity of the fluorescence emission fromthe samples. Samples of fish products are liquified and mixed with aniron broth growth medium forming an incubation mixture, and areincubated for an incubation period. The incubation mixture may beincubated, for example, at a temperature of 28° C.±0.5° C., 29° C.±0.5°C., 30° C.±0.5° C., 31° C.±0.5° C., 32° C.±0.5° C., 33° C.±0.5° C., 34°C.±0.5° C., or 35° C.±0.5° C.

Growth of SPB visibly alters the iron broth in two stages. In the firststage, the color of the iron broth changes from yellow to bright yellow(both transparent). In the second stage, iron sulfide precipitate (FeS)starts to form, causing the sample to become gray and eventually black(opaque). When measured with the fluorometer, the first visible stage(yellow) is expressed as an increase in fluorescence signal units andthe second visible stage (gray) is expressed as a reduction offluorescence signal units. The time at which a fluorescence peak occursrepresents the “time to detection” of SPB of the sample. The quantity ofSPB is related to the concentration of FeS. When the concentration ofFeS in the incubation mixture reaches a particular level, thefluorescent light emitted is reduced and the fluorescence begins todecrease, the fluorescence peak having been achieved. Fluorescence ismeasured at regular intervals, for example, every half-hour or hour.Typically, at least three fluorescence measurements are necessary toidentify when the fluorescence peak occurs.

Briefly, the methods described herein may be carried out by mixing a“stomached” sample (or alternatively a “non-stomached” sample) of a foodproduct with peptone water. A sample of the peptone water mixture isadded to a growth medium (iron broth media) to make an incubationmixture and incubating the incubation mixture at a predeterminedtemperature (e.g. 30° C.). The results (SPB concentration of the foodproduct) are determined by (1) detecting a visible color change in themixture from yellow to black, (2) manually taking a series offluorescence measurements of the incubation mixture, including duringthe visible color change from yellow to black, or (3) automaticallytaking a series of fluorescence measurements during the course ofincubation of the sample. The conventional method for detection andenumeration of sulfide-producing bacteria can be performed as areference if desired.

METHODS

Iron Broth Media

Iron agar Lyngby (IAL) is a conventional growth medium used in thedetermination of total viable counts (TVC) of H₂S-producing organismsand is made by combining 2% peptone (Difco); 0.3% Lab Lemco powder(Oxoid); 0.3% yeast extract (Difco); 0.03% ferric-citrate (Merck); 0.03%sodium thiosulfate (Merck); 0.5% NaCl (Merck); and 1.2% agar (Difco)dissolved in 1000 ml distilled water. The pH is then adjusted to 8.2. Todissolve the ferric-citrate completely, sterilization of the medium at121° C. for 15 minutes is required, obtaining a pH of 7.4±0.2. Aftersterilization, sterile-filtered L-cysteine; 0.04% (Sigma) is added.

In an alternate version of the iron broth medium, ferrous sulfate (Fe³⁺)could be substituted for ferric citrate (Fe²⁺) at the same concentration(0.03%). The alternate medium enables a color change from yellow to graywhich is detectable earlier due to a more prominent blackening of themedium.

For the methods of rapid detection of sulfide-producing bacteriadescribed herein, the samples are cultivated in iron broth (IAL mediaminus agar) rather than Iron Agar Lyngby. Peptone water is used fordilution of samples and is formed by dissolving 8.5 gm NaCl (Difco) and1 gm peptone (Difco) in 1000 ml of water. The peptone water is thensterilized at 121° C. for 15 min.

Preparation of Meat Sample

A sample of meat or fish (10 g) is homogenized in 30 ml of peptone waterin a stomacher filterbag (Seward, model 400 6041/str) using a stomacher(Lab Blender 400). Required time in the stomacher is approximately 4minutes or until the sample is homogenized. A subsample of 1.5 ml of thestomached sample is dispensed from the filterbag and added to a steriletube containing 4.5 ml of iron broth as described further herein. Thetube is capped and mixed. In an alternative embodiment the food samplemay be left intact and inserted into the medium. In one embodiment thefood sample is a 1 cm³ cube.

As noted, elsewhere herein the sample of the food product may comprise,for example, fish, shellfish, cheese, poultry, beef, pork, lamb, orother fish, meat or dairy products.

Fluorescence Measurement

In a preferred embodiment of the present invention, a laboratoryfluorometer is used to detect SPB. In this embodiment, a fluorometersuch as a TURNER DESIGNS TD-700 fluorometer can be used. The TD-700 hastwo optical filters: an excitation filter with a 380 nm narrow bandpass, and an emission filter with a 450 nm narrow band pass. The TD-700is single point calibrated using pure iron broth media for setting theoptimal sensitivity and range for the fluorometer. Pure iron broth mediais set to 400 fluorescence units (fsu). Fluorescence measurements inthis embodiment preferably are taken each 15-30 minutes and preferablyare incubated at 30° C. In another embodiment, fluorescence isautomatically measured using a COLIFAST CA-100.

Standard Fluorescence Curve

To prepare a standard curve for use in the fluorescence test, S.putrefaciens can be grown overnight in iron broth at 20° C. After thispre-enrichment step, serial 10-fold dilutions are made using peptonewater. A sample of the peptone water is transferred to iron broth andmixed. From each dilution of the iron broth mixture, two parallelsamples of 3 ml are transferred to two cuvettes. The samples arepreferably incubated in the open cuvettes at 30° C. Before using thefluorometer to measure fluorescence, the cuvette is capped and mixed byinversion in order to obtain a homogenous sample. The fluorescence isgenerally measured at room temperature (18° C.-20° C.).

EXAMPLES

Referring now to FIG. 1, the results for rapid enumeration of S.putrefaciens in pure cell culture cultivated in iron broth are shown.FIG. 1 shows the relationship between fluorescence and concentration ofbacteria with regard to the time of detection. When bacterialconcentrations are initially high, the “time to detection” (TTD) or“detection time” is very early in the incubation period, whereas, wheninitial concentration is low, “time to detection” is much later in theincubation period.

Referring to FIG. 2, the graph shows the development of fluorescence insamples of cod contaminated with varying levels of S. putrefaciens. Thesamples are cultivated in iron broth and incubated at 30° C. As bacteriaincrease in each sample, there is an increase in fluorescence over aperiod of time which is indicated by the upward curve in the graph. Whenthe fluorescence begins to decrease as more iron sulfide is formed, apeak has been formed. In FIG. 2, the cod sample having about 10⁴ cfu/gof S. putrefaciens attains a fluorescence peak (i.e. time to detection)in about 8 hours, for example.

Referring now to FIG. 3, the graph therein shows the correlation betweenthe time to detection and the number of sulfide-producing bacteria insamples of several fish species. As shown in FIG. 3, there is a strongnegative correlation (high R²) between the number of sulfide-producingbacteria in a sample, and the time necessary to detect the bacteria in asample of contaminated food, fish or meat, where time to detection isdefined as the time required to achieve maximum (peak) fluorescence.

Though not wishing to be constrained by theory, it is suspected that theincrease in fluorescence can be explained by the decrease in theconcentration of Fe³⁺ in the broth due to the formation of Fe²⁺. When aspecific concentration of sulfide-producing bacteria is reached in themedium, the fluorescence decreases. This is visually observed as aconversion from yellow to a gray color in the incubation mixture whicheventually turns black due to FeS formation. The detection time (thex-axis in FIG. 3) is defined as the distance from the y-axis (start ofanalysis) to the point of maximum (peak) fluorescence.

In the invention contemplated herein, a method of detecting andenumerating SPB in a food sample, as defined elsewhere herein, includespreparing the food sample, for example a fish sample as a liquified foodsample. Fish or shellfish, for example may be selected from the groupcomprising fish (including cod, coal fish, wolf fish, red fish, haddock,and salmon), molluscs, marine animals, crustaceans, or any productderived therefrom. An incubation mixture is formed by combining theliquified food sample with a growth medium comprising an iron compoundand a sulfur compound. An iron precipitate is formed in the incubationmixture when SPB act upon the iron compound and sulfur compound in thegrowth medium. The incubation mixture is incubated at an incubationtemperature for an incubation period. The incubation temperature of themethod is preferably from about 28° C.±0.5° C. to about 35° C.±0.5° C.and more preferably about 30° C.±0.5° C. The incubation period isgenerally from about 3 to about 17 hours and may be any incremental timeperiod therein. A plurality of fluorescence measurements (usually atleast three) are taken from the incubation mixture during the incubationperiod. It is concluded that the food sample contains a particularquantity of SPB when the fluorescence measurements taken from theincubation mixture show a maximum fluorescence. The measurementsindicate a fluorescence peak in the incubation mixture during theincubation period (an increase followed by a decrease).

Enumeration of SPB by Fluorescence Detection

In an especially preferred embodiment of the invention, SPB in food,particularly fish, products are detected and enumerated using manual orautomatic fluorescence measurement techniques.

Portions of an incubation mixture containing the stomached fish or fish(or food) product are incubated at a predetermined temperature,preferably 30° C.±0.5° C. and fluorescence measurements are takenmanually or automatically at predetermined intervals, for example, every15, 30, 45 or 60 minutes. Fluorescence measurements are continued to betaken at appropriate intervals at least until a peak (a maximum) in thelevel of fluorescence is detected. As noted earlier, the time until thefluorescence peak is attained is referred to as the “time to detection”(TTD) or “detection time”. TTD generally occurs during the phase ofincubation when the visible color of the incubation mixture changes fromyellow to black. Therefore, optimally, fluorescence measurements areespecially preferred to be taken during the yellow-to-black color changephase to ensure that the fluorescence peak (the fluorescence maximum) isdetected.

As noted previously, the results shown in FIGS. 1 and 2 indicate thatwhen concentrations of SPB are initially high, the TTD occurs earlyduring the incubation period, whereas when concentrations of SPB arelow, the TTD occurs much later during the incubation period.

TTD can be used to provide a quantitative enumeration of SPB in theoriginal sample of food or fish or fish product by comparing the TTD fora particular product to a previously formulated correlation schedule,such as shown in FIG. 3 for a variety of fish species including cod,coal fish, wolf fish and salmon. It is well within the ability of aperson of ordinary skill in the art, given the teachings herein, toformulate a correlation schedule such as FIG. 3 or Table 1 which relatestime to peak fluorescence (TTD) to concentration of SPB in the product.

In one version of the invention for example, when the TTD is withinabout 10±0.5 hours, a fish sample is considered to have about 10⁴ CFU/gof SPB or more (Table 1). A level of 10⁶ CFU/g is generally consideredto be the maximum level of SPB which can be present in a fish sample tobe shipped in a fresh condition. When the TTD is within about 6±0.5hours, the fish sample is considered to have at least 10⁶ CFU/g. Whenthe TTD is greater than 6±0.5 hours, the fish sample is considered tohave less than 10⁶ CFU/g.

Any such TTD or CFU/g threshold can be used in a “pass-fail” version ofthe present invention (for example, using the values in Table 1). Forexample, if the threshold is 10⁴ CFU/g, then the fish sample “fails”(i.e., has at least 10⁴ CFU/g) if the TTD is less than about 10±0.5hours, and “passes” (i.e., has fewer than 10⁴ CFU/g) if the TTD isgreater than about 10±0.5 hours. Similarly, if the threshold is 10⁶CFU/g, then the fish sample “fails” (i.e., has at least 10⁶ CFU/g) ifthe TTD is less than about 6±0.5 hours, and “passes” (i.e., has fewerthan 10⁶ CFU/g) if the TTD is greater than about 6±0.5 hours.“Pass-fail” thresholds which have more narrow TTD ranges can be easilycalculated for each testing site based on specific types of foodproducts desired to be tested and testing procedures and facilities ateach site.

TABLE 1 Correlation of Detection Time (TTD) and Numbers ofSulfide-Producing Bacteria (CFU/g) Detection Time (hr) Number of SPB(CFU/g)  4 ± .5 10⁷  5 ± .5 5 × 10⁶  6 ± .5 10⁶  7 ± .5 5 × 10⁵  8 ± .510⁵  9 ± .5 5 × 10⁴ 10 ± .5 10⁴ 11 ± .5 5 × 10³ 12 ± .5 10³ 13 ± .5 10²14 ± .5 5 × 10¹Visual Detection and Enumeration

In a preferred embodiment of the present invention, sulfide-producingbacteria are detected and semi-quantitatively enumerated visually (usingchanges in visible light) rather than by fluorescence detection. In oneembodiment, a sample of food or fish (stomached or intact) is combinedwith a sterile iron broth as described above to form a sample-mediamixture (an incubation mixture). The incubation mixture is thenincubated at a predetermined incubation temperature such as 30° C., oranother temperature as described elsewhere herein.

The color of the incubation mixture is assessed visually at intervalsduring the incubation period to determine at what time the samplechanges color from yellow to black (FIG. 4). The color of the incubationmixture at any particular incubation time can be matched with acorrelation schedule such as a detection chart (Table 2, for example) tomake a semi-quantitative and quality determination of the concentrationof sulfide-producing bacteria in the food sample at that time interval.

For example, using Table 2, if the incubation mixture is still yellowafter about 3-6 hours of incubation, the original sample is consideredto have less than about 5×10⁶ CFU/gm. If the color of the incubationmixture has changed from yellow to black after 3-6 hours, the originalsample is considered to have at least about 5×10⁶ CFU/gm wherein theoriginal sample is considered to have “bad” quality.

If the color of the incubation mixture is still yellow after from about7-10 hours of incubation, the sample is considered to have less thanabout 5×10⁵ CFU/gm. If the color of the incubation mixture has changedfrom yellow to black after from about 7-10 hours of incubation, thesample is considered to have at least about 5×10⁵ CFU/gm to 5×10⁶CFU/gm. Wherein the original sample is considered to have “marginal”quality.

If the color of the incubation mixture is still yellow after from about11-12 hours of incubation, the sample is considered to have less thanabout 10³ CFU/gm. If the color of the incubation mixture has changedfrom yellow to black after from about 11-12 hours of incubation, thesample is considered to have at least about 10³ to 5×10⁵ CFU/gm whereinthe original sample is considered to have “good” quality.

If the color of the incubation mixture has changed from yellow to blackonly after at least twelve hours of incubation, the sample is consideredto have less than about 10³ CFU/gm wherein the original sample isconsidered to have “very good” quality.

It will be understood by those of ordinary skill in the art that whenother conditions are used to incubate the incubation mixtures, forexample using other incubation temperatures, or using other growthmedia, the concentrations and incubation times listed in Table 2 may beadjusted and the determination of such adjustments are considered to bewell within the ability of a person of ordinary skill in the art usingstandard techniques.

TABLE 2 Semi-Quantitative Enumeration of Sulfide-Producing BacteriaApproximate CFU/g for samples incubated in iron broth at 30° C.INCUBATION TIME (Hours until sample turns Black) (hr) SPB (CFU/g) 3-6 >5× 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³ − 5 × 10⁵ >12 <10³

In one embodiment of the visual detection and enumeration method, aportion of the food product sample to be tested is removed intacttherefrom and is placed directly (without stomaching) into a containerwith the iron broth to form an incubation mixture. For example, for eachintact 2-gram portion or 1c³ portion, about 7 ml of iron broth medium isused. The medium with the intact portion is incubated in the same manneras the visual detection method described above. The color of theincubation mixture of the intact portion is observed at intervals asdescribed elsewhere herein. The incubation time required to observe achange of the color of the medium from yellow to black is then matchedwith a correlation schedule such as the detection chart of Table 2 tomake a semi-quantitative determination or “quality” determination of theSPB in the original food sample as described above.

In this method wherein the food sample is an intact fish sample, thefish sample should have at least one surface which has been in contactwith air and does not contain skin. When placed in the medium the sampleshould be covered by the medium. The vial containing the incubationmixture should be immediately placed in an incubator in an uprightposition. No disinfectants should have been used on the sample, beforeor after removal from the fish product. Fresh fish rather than salted,smoked, or preserved fish are used preferably.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims.

1. A method of detecting and/or enumerating sulfide-producing bacteriain a fish sample, comprising: preparing the fish sample as a liquifiedfish sample; forming an incubation mixture by combining the liquifiedfish sample with a growth medium comprising iron and sulfur, and whereinan iron precipitate is formed in the incubation mixture whensulfide-producing bacteria act upon the iron and sulfur in the growthmedium; incubating the incubation mixture at an incubation temperature;taking a plurality of fluorescence measurements from the incubationmixture during the incubation period to detect a fluorescence peak, thefluorescence peak comprising a fluorescence increase followed by afluorescence decrease, the fluorescence decrease coinciding with anaccumulation of the iron precipitate in the incubation mixture, whereinthe period of time to the fluorescence peak comprises a detection time;and estimating the number of sulfide-producing bacteria in the fishsample by comparing the detection time to a correlation schedule.
 2. Themethod of claim 1 wherein in the step of incubating the incubationmixture, the incubation temperature is from about 28° C.±0.5° C. toabout 35° C.±0.5° C.
 3. The method of claim 1 wherein in the step ofincubating the incubation mixture, the incubation temperature is about30° C.±0.5° C.
 4. The method of claim 1 wherein in the step ofincubating the incubation mixture, the incubation period is from about 3hours to about 17 hours.
 5. The method of claim 1 wherein in the step offorming an incubation mixture, the iron is provided as ferric citrate orferrous sulfate.
 6. The method of claim 1 wherein in the step of formingan incubation mixture, the sulfur is provided as cysteine andthiosulfate.
 7. The method of claim 1 wherein in the step of taking aplurality of fluorescence measurements, the fluorescence measurementsare taken manually.
 8. The method of claim 1 wherein in the step oftaking a plurality of fluorescence measurements, the fluorescencemeasurements are taken automatically.
 9. The method of claim 1 whereinin the step of estimating the number of sulfide-producing bacteria, thecorrelation schedule comprises: Detection Time (hr) Number of SPB(CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³⁻ 5 × 10⁵ >12 <10³.


10. A method of detecting and/or enumerating sulfide-producing bacteriain a fish sample, comprising: preparing the fish sample as a liquifiedfish sample; forming an incubation mixture by combining the liquifiedfish sample with a growth medium comprising iron and sulfur, and whereinan iron precipitate is formed in the incubation mixture whensulfide-producing bacteria act upon the iron and the sulfur in thegrowth medium; incubating the incubation mixture at an incubationtemperature for a predetermined incubation period which produces afluorescence peak when a particular number of sulfide-producing bacteriaare present; taking a plurality of fluorescence measurements from theincubation mixture during the predetermined incubation period; andconcluding that the fish sample has at least the particular number ofsulfide-producing bacteria when a fluorescence peak is attained by theend of the predetermined incubation period, and that the fish sampledoes not have the predetermined number of sulfide-producing bacteriawhen a fluorescence peak is not attained by the end of the predeterminedincubation period.
 11. The method of claim 10 wherein in the step ofincubating the incubation mixture, the incubation temperature is fromabout 28° C.±0.5° C. to about 35° C.±0.5° C.
 12. The method of claim 10wherein in the step of incubating the incubation mixture, the incubationtemperature is about 30° C.±0.5° C.
 13. The method of claim 10 whereinin the step of incubating the incubation mixture, the predeterminedincubation period is one of 3±0.5 hours, 4±0.5 hours, 5±0.5 hours, 6±0.5hours, 7±0.5 hours, 8±0.5 hours, 9±0.5 hours, 10±0.5 hours, 11±0.5hours, 12±0.5 hours, 13±0.5 hours and 14±0.5 hours.
 14. The method ofclaim 10 wherein in the step of incubating the incubation mixture, theparticular number of sulfide-producing bacteria is one of 10² CFU/g, 10³CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 5×10⁵ CFU/g, 10⁶ CFU/g, 10⁶ CFU/g, 10⁷CFU/g and 10⁸ CFU/g.
 15. The method of claim 10 wherein in the step offorming an incubation mixture, the iron is provided as ferric citrate orferrous sulfate.
 16. The method of claim 10 wherein in the step offorming an incubation mixture, the sulfur is provided as cysteine andthiosulfate.
 17. The method of claim 10 wherein in the step of taking aplurality of fluorescence measurements, the fluorescence measurementsare taken manually.
 18. The method of claim 10 wherein in the step oftaking a plurality of fluorescence measurements, the fluorescencemeasurements are taken automatically.
 19. A method of detecting orenumerating sulfide-producing bacteria in a fish sample, comprising:preparing the fish sample as a liquified fish sample; forming anincubation mixture by combining the liquified fish sample with a growthmedium having a color and comprising iron and sulfur, and wherein aniron precipitate is formed in the incubation mixture whensulfide-producing bacteria act upon the iron and sulfur in the growthmedium; incubating the incubation mixture at an incubation temperature;determining a time to color change at which time the color of theincubation mixture changes from yellow to black due to an accumulationof the iron precipitate in the incubation mixture; and estimating thenumber of sulfide-producing bacteria in the fish sample by comparing thetime to color change to a correlation schedule.
 20. The method of claim19 wherein in the step of incubating the incubation mixture, theincubation temperature is from about 28° C.±0.5° C. to about 35° C.±0.5°C.
 21. The method of claim 19 wherein in the step of incubating theincubation mixture, the incubation temperature is about 30° C.±0.5° C.22. The method of claim 19 wherein in the step of incubating theincubation mixture, the predetermined incubation period is one of 3±0.5hours, 4±0.5 hours, 5±0.5 hours, 6±0.5 hours, 7±0.5 hours, 8±0.5 hours,9±0.5 hours, 10±0.5 hours, 11±0.5 hours, 12±0.5 hours, 13±0.5 hours and14±0.5 hours.
 23. The method of claim 19 wherein in the step ofincubating the incubation mixture, the number of sulfide-producingbacteria is one of 10² CFU/g, 10³ CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 5×10⁵CFU/g, 10⁶ CFU/g, 5×10⁶ CFU/g, 10⁷ CFU/g and 10⁸ CFU/g.
 24. The methodof claim 19 wherein in the step of forming an incubation mixture, theiron is provided as ferric citrate or ferrous sulfate.
 25. The method ofclaim 19 wherein in the step of forming an incubation mixture, thesulfur is provided as cysteine and thiosulfate.
 26. The method of claim19 wherein in the step of estimating the number of sulfide-producingbacteria, the correlation schedule comprises: Detection Time (hr) Numberof SPB (CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³⁻ 5 ×10⁵ >12 <10³.


27. A method of detecting and/or enumerating sulfide-producing bacteriain a food sample, comprising: providing a food sample; providing agrowth medium comprising iron and sulfur and disposing the food sampleinto the growth medium forming an incubation mixture, and wherein aniron precipitate is formed in the incubation mixture whensulfide-producing bacteria from the food sample act upon the iron andsulfur in the growth medium; incubating the incubation mixture at anincubation temperature; determining a time to color change at which timethe color of the incubation mixture changes to black due to anaccumulation of the iron precipitate in the incubation mixture; andestimating the number of sulfide-producing bacteria on the food sampleby comparing the time to color change to a correlation schedule.
 28. Themethod of claim 27 wherein in the step of providing the food sample, thefood sample is selected from the group comprising beef, lamb, pork,poultry, fish, shellfish, molluscs, marine animals, crustaceans, or anyproduct derived therefrom.
 29. The method of claim 27 wherein the foodsample is an intact food sample.
 30. The method of claim 27 wherein inthe step of incubating the incubation mixture, the incubationtemperature is from about 28° C.±0.5° C. to about 35° C.±0.5° C.
 31. Themethod of claim 27 wherein in the step of incubating the incubationmixture, the incubation temperature is about 30° C.±0.5° C.
 32. Themethod of claim 27 wherein in the step of incubating the incubationmixture, the predetermined incubation period is one of 3±0.5 hours,4±0.5 hours, 5±0.5 hours, 6±0.5 hours, 7±0.5 hours, 8±0.5 hours, 9±0.5hours, 10±0.5 hours, 11±0.5 hours, 12±0.5 hours, 13±0.5 hours and 14±0.5hours.
 33. The method of claim 27 wherein in the step of incubating theincubation mixture, the particular number of sulfide-producing bacteriais one of 10² CFU/g, 10³ CFU/g, 10⁴ CFU/g, 10⁵ CFU/g, 5×10⁵ CFU/g, 10⁶CFU/g, 5×10⁶ CFU/g, 10⁷ CFU/g and 10⁸ CFU/g.
 34. The method of claim 27wherein in the step of forming an incubation mixture, the iron isprovided as ferric citrate or ferrous sulfate.
 35. The method of claim27 wherein in the step of forming an incubation mixture, the sulfur isprovided as cysteine and thiosulfate.
 36. The method of claim 27 whereinin the step of estimating the number of sulfide-producing bacteria, thecorrelation schedule comprises: Detection Time (hr) Number of SPB(CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5 × 10⁶ 11-12 10³⁻ 5 × 10⁵ >12 <10³.


37. A method of detecting and/or enumerating sulfide-producing bacteriain a fish sample, comprising: providing a fish sample; providing agrowth medium comprising iron and sulfur and disposing the fish sampleinto the growth medium forming an incubation mixture, and wherein aniron precipitate is formed in the incubation mixture whensulfide-producing bacteria from the fish sample act upon the iron andsulfur in the growth medium; incubating the incubation mixture at anincubation temperature; determining a time to color change at which timethe color of the incubation mixture changes to black due to anaccumulation of the iron precipitate in the incubation mixture; andestimating the number of sulfide-producing bacteria on the fish sampleby comparing the time to color change to a correlation schedule.
 38. Themethod of claim 37 wherein in the step of providing the fish sample, thefish sample is selected from the group comprising freshwater fish,saltwater fish, shellfish, and molluscs, and crustaceans, or any productderived therefrom.
 39. The method of claim 37 wherein the fish sample isan intact fish sample.
 40. The method of claim 37 wherein in the step ofincubating the incubation mixture, the incubation temperature is fromabout 28° C.±0.5° C. to about 35° C.±0.5° C.
 41. The method of claim 37wherein in the step of incubating the incubation mixture, the incubationtemperature is about 30° C.±0.5° C.
 42. The method of claim 37 whereinin the step of incubating the incubation mixture, the predeterminedincubation period is one of 3±0.5 hours, 4±0.5 hours, 5±0.5 hours, 6±0.5hours, 7±0.5 hours, 8±0.5 hours, 9±0.5 hours, 10±0.5 hours, 11±0.5hours, 12±0.5 hours, 13±0.5 hours and 14±0.5 hours.
 43. The method ofclaim 37 wherein in the step of incubating the incubation mixture, thenumber of sulfide-producing bacteria is one of 10² CFU/g, 10³ CFU/g, 10⁴CFU/g, 10⁵ CFU/g, 5×10⁵ CFU/g, 10⁶ CFU/g, 5×10⁶ CFU/g, 10⁷ CFU/g and 10⁸CFU/g.
 44. The method of claim 37 wherein in the step of forming anincubation mixture, the iron is provided as ferric citrate or ferroussulfate.
 45. The method of claim 37 wherein in the step of forming anincubation mixture, the sulfur is provided as cysteine and thiosulfate.46. The method of claim 37 wherein in the step of estimating the numberof sulfide-producing bacteria, the correlation schedule comprises:Detection Time (hr) Number of SPB (CFU/g) 3-6 5 × 10⁶  7-10 5 × 10⁵ − 5× 10⁶ 11-12 10³⁻ 5 × 10⁵ >12 <10³.